1. Technical Field
The present invention relates to a method for producing L-lysine or L-threonine using an Escherichia bacterium. L-Lysine and L-threonine are known as essential amino acids, and are useful as components in pharmaceutical compositions and diverse nutritive mixtures, such as additives to animal fodder.
2. Background Art
L-amino acids, such as L-threonine and L-lysine, are industrially produced by fermentation using L-amino acid-producing bacteria, such as coryneform or Escherichia bacteria which have the ability to produce L-amino acids. To improve productivity, a strain isolated from nature, an artificial mutant thereof, or a recombinant in which the L-amino acid biosynthetic enzyme activity is increased by gene recombination has been used as the L-amino acid-producing bacterium. The method for producing L-lysine is exemplified in Japanese Patent Application Laid-Open No. 10-165180, Japanese Patent Application Laid-Open No. 11-192088, Japanese Patent Application Laid-Open No. 2000-253879, and Japanese Patent Application Laid-Open No. 2001-57896. The method for producing L-threonine is exemplified in Japanese Patent Application Laid-Open No. 5-304969, International Publication No. WO98/04715, Japanese Patent Application Laid-Open No. 5-227977, and U.S. Patent Application Publication No. 2002/0110876.
Methods for increasing the ability to produce amino acids such as L-threonine and L-lysine include a method for increasing energy efficiency by modifying a respiratory chain pathway (Japanese Patent Application Laid-Open No. 2002-17363), and a method for increasing an ability to produce nicotinamide adenine dinucleotide phosphate by amplifying a nicotinamide nucleotide transdehydrogenase (Japanese Patent No. 2817400), as well as a method of increasing an expression amount of an enzyme of the endogenous biosynthetic pathway.
In addition, methods for modifying common pathways of amino acid biosynthetic systems are known and include modifying anaplerotic pathways of L-amino acid-producing bacteria, such as an L-lysine-producing coryneform bacterium in which pyruvate carboxylase activity is increased (Japanese Patent Application Laid-Open No. 2002-508921), an L-lysine-producing Escherichia bacterium which is deficient in pyruvate kinase (International Publication No. WO03/008600), and an L-lysine-producing coryneform bacterium which is deficient in malate quinine oxidoreductase (U.S. Patent Application Publication No. 2003/0044943).
A malic enzyme is one of the anaplerotic pathway enzymes. In the Escherichia bacteria, it is known that each of the sfcA and b2463 genes encodes the malic enzyme (van der Rest, M. E., Frank C., Molenaar, D. J., J. Bacteriol., 182(24):6892-6899, 2000). However, whether or not a decrease in the activity of the malic enzymes encoded by the sfcA and b2463 genes is effective for enhancing L-lysine or L-threonine production has not been reported.
A metabolic flux analysis, which is also referred to as a flux balance analysis, is a technique for predicting intracellular metabolic flux distributions by construction of a stoichiometric model of intracellular biochemical reactions and linear optimization. This technique has been used in research into the abilities of biochemical reaction systems in microorganisms or for predicting intracellular metabolic flux distributions under different external conditions (Varma, A. and Palsson, B. O., Appl. Environ. Microbiol. 60:3724-3731, 1994, Schilling, C. H., et al., Biotechnol. Prog., 15:288-295, 1999, and Schilling, C. H., et al., Biotechnol. Prog., 15:296-303, 1999). It has also been reported that a stoichiometric model was constructed for Escherichia coli (Pramanik, J. and Keasling, J. D., Biotechnol. Bioeng., 56:398-421, 1997, and Ibarra, R. U., et al., Nature, 420:186-189, 2002). Also known is an example of using such a stoichiometric model in metabolic engineering for lysine production for Corynebacterium glutamicum, which is used in amino acid production (Vallino, J. J. and Stephanopoulos, G., Biotechnol. Bioeng., 41:633-646, 1993). In addition, a large number of theoretical or experimental methods for metabolic flux analyses and their applications have been reported (Wiechert, W., Journal of Biotechnology, 94:37-63, 2002, Wiechert, W., Metabolic Engineering, 3:195-205, 2001, International Publication No. WO00/46405, International Publication No. WO02/061115, and International Publication No. WO02/055995). International Publication No. WO00/46405 discloses a method for predicting a gene required for growth based on a stoichiometric model. International Publication No. WO02/061115 discloses a technique for genetically and evolutionarily changing cells to impart optimal functions to the cells. Furthermore, International Publication No. WO02/055995 discloses a method for applying limitations of qualitative kinetic information, limitations of qualitative control information, and limitations based on DNA microarray experimental data under different conditions, to a stoichiometric model. Although all of these are methods for predicting more desirable intracellular metabolic flux distributions, no method has been disclosed for theoretically predicting a specific flux as a target for directly improving cellular substance production.